A. Field of the Invention
The present invention is related generally to the detection of protein in biological samples; and more particularly, to a novel method for the determination of protein in a biological sample using novel protein error indicators.
B. Description of the Background Art
Determining the presence of protein in a biological sample is of utmost importance in the diagnosis of several pathological conditions affecting the kidney, circulatory system, and central nervous system. Frequently, it is necessary to qualitatively and quantitatively measure protein (albumin) in urine. This is especially important in the diagnosis of diabetes, urinary tract infection, and kidney disease. The predominant protein in diabetes and kidney disease is albumin; hence the model system for protein urine testing is albumin.
Methods for determining the presence of albumin in urine are well known. The most inexpensive and convenient method for albumin determination involves wetting a paper test strip with a small quantity of urine. The test strip is impregnated with a protein error indicator. If albumin is present in the sample, the test strip will indicate this by simply changing color. .The color observed may vary depending on the concentration of albumin in the sample. This variable color change is used to quantify the albumin in the sample. Test papers of the above-type require a minimum of training to use correctly. These test strips provide an accurate, convenient, and rapid vehicle for the on-the-spot determination of protein. Test papers such as these are widely used by technicians in clinical laboratories, as well as by physicians in their offices.
In more detail, these test strips include an absorbent carrier strip, i.e., paper, impregnated with a buffer, a polymer/surfactant (required for stability, wettability or to prevent leaching of the buffer) and a protein error indicator. Substantially all protein error indicators used in commercial dry phase tests are triphenylmethane derivatives sharing the basic structures below: ##STR1##
Structure A represents the general structure of triphenylmethane derivatives in protic solvents (water, alcohols, etc.) while structure B represents the form that predominates in the dry state or in aprotic solvents (ethers, acetonitrile, etc). Generally, triphenylmethane derived protein error indicators (phenolsulfonephthaleins) are represented as structure B. For purposes of consistency the protein error indicators of the present invention will be represented using structure B. It should be understood, however, that the protein error indicators of the present invention can also exist as structure A.
Protein error indicators are pH indicators including an ionizable group which has a pKa value that is displaced by the presence of protein. In the case of phenolsulfonephthaleins, the ionizable group is the C ring phenolic hydroxyl. The pKa value of a phenolsulfonephthalein indicator is the pH value at which one-half of the number of indicator molecules include deprotonated C ring phenolic hydroxy groups.
With regard to the phenolsulfonephthalein protein error indicators illustrated above, two deprotonation events occur in order to cause an observable color change. The first deprotonation removes the proton from the aryl sulfonic acid to yield the compound illustrated below: ##STR2## The pKa of this proton is less than one. Thus, this moiety is ionized at all useful pH values. This ionized group is also responsible for the aqueous solubility of these compounds.
The second deprotonation involves releasing a proton from the C ring phenolic hydroxyl to yield the dianion below: ##STR3## In protein error indicators, the second deprotonation causes the observable color change which is indicative of protein in the sample being tested.
The buffer provides the indicator an environment of constant pH in which to function. Thus, when the test strip is dipped into a biological fluid, which often has a significantly different pH value from the buffered environment, the indicator is not influenced by the pH of the biological fluid. This ensures that any subsequent color change in the indicator is a result of a shift in the indicator's pKa value and not a result of the pH of the sample being tested.
Test strips which are generally considered useful for the analytical determination of protein in a biological sample are described in U.S. Pat. Nos. 2,986,453, 3,095,277, 3,485,587 and 4,013,416. The test strips described therein include an absorbent carrier impregnated with a water immiscible polypropylene glycol, a buffer, and a pH indicator of the octahalosulfophthalein group. The octahalosulfophthalein indicators are triphenylmethane derivatives halogenated at the 3', 3", 5', 5", 3, 4, 5, and 6 positions. According to the patent, test strips including octahalosulfo-phthalein and water immiscible polypropylene glycols are disturbed less by interfering nitrogen-containing compounds in the test sample than test strips including other phenolsulfonephthalein indicators and surface active agents.
Although the test strips described above are less disturbed by nitrogen-containing compounds in the sample, they and other presently available test strips suffer from several common serious disadvantages. Presently available test strips have strong background negative coloration. For example, the indicators of the octahalosulfophthalein group are yellow colored in the absence of albumin. Subsequently, when albumin is added to the sample, the color changes from yellow to yellow-green to green, depending on the concentration of albumin in the sample. This background coloration is especially troublesome when it is considered that the biological fluid most often tested is urine which is normally colored yellow. Thus, small changes in the color of the test strip caused by trace amounts of albumin, i.e., from about 10 to 30 mg/dl (milligrams per deciliter) in urine could easily be masked by the color of the sample itself and go undetected. This problem is further compounded since these test strips are used by minimally trained technicians who may experience increased difficulty in interpreting the observed results. Because medical treatment is often initiated based on the results of these tests, the accurate interpretation of the results is imperative. Further, presently available test strips are not sensitive enough to detect very low levels of protein. Urinary albumin levels of from about 3 to about 10 mg/dl are significant in diagnosing several life threatening pathologies, such as diabetes and kidney disease. Nevertheless, test strips presently available cannot always accurately detect albumin below about 10 to 15 mg/dl.
Accordingly, to overcome the shortcomings discussed above, it would be extremely advantageous to provide a protein error indicator which changes from no color (colorless) to color in the presence of albumin. It would be even more advantageous if the protein error indicator accurately and clearly indicated whether albumin was present at concentrations below those presently detectable. A still further advantage would be realized by providing a test strip including such a protein error indicator.